Shear-induced Platelet Aggregation Research Articles

Influence of Hematocrit Level and Integrin αIIbβIII Function on vWF-Mediated Platelet Adhesion and Shear-Induced Platelet Aggregation in a Sudden Expansion.

Shear-mediated thrombosis is a clinically relevant phenomenon that underlies excessive arterial thrombosis and device-induced thrombosis. Red blood cells are known to mechanically contribute to physiological hemostasis through margination of platelets and vWF, facilitating the unfurling of vWF multimers, and increasing the fraction of thrombus-contacting platelets. Shear also plays a role in this phenomenon, increasing both the degree of margination and the near-wall forces experienced by vWF and platelets leading to unfurling and activation. Despite this, the contribution of red blood cells in shear-induced platelet aggregation has not been fully investigated-specifically the effect of elevated hematocrit has not yet been demonstrated. Here, a microfluidic model of a sudden expansion is presented as a platform for investigating platelet adhesion at hematocrits ranging from 0 to 60% and shear rates ranging from 1000 to 10,000 s-1. The sudden expansion geometry models nonphysiological flow separation characteristic to mechanical circulatory support devices, and the validatory framework of the FDA benchmark nozzle. PDMS microchannels were fabricated and coated with human collagen. Platelets were fluorescently tagged, and blood was reconstituted at variable hematocrit prior to perfusion experiments. Integrin function of selected blood samples was inhibited by a blocking antibody, and platelet adhesion and aggregation over the course of perfusion was monitored. Increasing shear rates at physiological and elevated hematocrit levels facilitate robust platelet adhesion and formation of large aggregates. Shear-induced platelet aggregation is demonstrated to be dependent on both αIIbβIII function and the presence of red blood cells. Inhibition of αIIbβIII results in an 86.4% reduction in overall platelet adhesion and an 85.7% reduction in thrombus size at 20-60% hematocrit. Hematocrit levels of 20% are inadequate for effective platelet margination and subsequent vWF tethering, resulting in notable decreases in platelet adhesion at 5000 and 10,000 s-1 compared to 40% and 60%. Inhibition of αIIbβIII triggered dramatic reductions in overall thrombus coverage and large aggregate formation. Stability of platelets tethered by vWF are demonstrated to be αIIbβIII-dependent, as adhesion of single platelets treated with A2A9, an anti-αIIbβIII blocking antibody, is transient and did not lead to sustained thrombus formation. This study highlights driving factors in vWF-mediated platelet adhesion that are relevant to clinical suppression of shear-induced thrombosis and in vitro assays of platelet adhesion. Primarily, increasing hematocrit promotes platelet margination, permitting shear-induced platelet aggregation through αIIbβIII-mediated adhesion at supraphysiological shear rates. The online version contains supplementary material available at 10.1007/s12195-024-00796-0.

Abstract 14010: Synergistic Influence of Rivaroxaban on Platelets, Inflammation and Coagulation Biomarkers in Patients With CAD and/or PAD on Aspirin Therapy

Background: Cardiovascular benefits of 2.5 mg twice daily rivaroxaban plus aspirin therapy (RIV+ASA) has been demonstrated in patients with arterial diseases. Hypothesis: RIV+ASA is associated with reduced platelet activation and plasma inflammation and coagulation activation markers in patients with CAD and/or PAD who were on ASA. Methods: In this open-label biomarker study, patients on 81 mg/day ASA were randomized to continue ASA or RIV+ASA for 12 weeks. We assessed ADP-, α-thrombin-, and tissue factor-induced platelet aggregation (PA) using conventional aggregometry, platelet-fibrin clot strength (PFCS) by INTEM (intrinsic pathway activator) and EXTEM (extrinsic pathway activator) using thromboelastometry, shear-induced PA by platelet function analyzer-100, D-dimer and fibrinogen using coagulation analyzer, hs-CRP and interleukin-6 using ELISA method at baseline, and four weeks and 12 weeks post-randomization. Results: Data was available in 9 patients with RIV + ASA and ten patients with ASA-only therapy. Most patients were male, Caucasians, obese and older. There were no differences in baseline demographics, medications, and laboratory values between groups, except patients in RIV + ASA group had higher white blood cell counts (p=0.028) and lower baseline 2uM ADP-induced PA (p=0.03). There were no differences in other laboratory measurements between baseline and post-randomization time points within the group or at 4- and 12-week time points between groups, except D-dimer values were significantly lower at 12 weeks (p=0.038) in the RIV + ASA vs. ASA-only group. No significant adverse events were observed. Conclusions: Twelve weeks of rivaroxaban plus aspirin vs. aspirin was associated with similar levels of platelet aggregation, platelet fibrin clot strength, fibrinogen and inflammation markers, and lower d-dimer levels.

Cordycepin Inhibits Pathological High Shear-Induced Platelet Aggregation, Activation, and Phosphatidylserine Exposure by Regulating the Phosphorylation of Akt Protein

Cordycepin Inhibits Pathological High Shear-Induced Platelet Aggregation, Activation, and Phosphatidylserine Exposure by Regulating the Phosphorylation of Akt Protein

Novel Antiplatelet Activity of Ginsenoside Re Through the Inhibition of High Shear Stress-Induced Platelet Aggregation.

Bleeding is one of the most serious side effects of antiplatelet drugs. Efforts have been made to find new antiplatelet agents without bleeding complications. Shear-induced platelet aggregation (SIPA) occurs only under pathological conditions and is a promising target for overcoming bleeding problems. This work demonstrates that the ginsenoside Re selectively inhibits platelet aggregation induced by high shear stress. Human platelets were exposed to high shear stress using microfluidic chip technology, and aggregation, activation, and phosphatidylserine (PS) exposure were measured. The Von Willebrand Ristocetin Cofactor (vWF:RCo) assay and western blot were used to evaluate the effect of the vWF-GPⅠb/PI3K/Akt signal pathway. The coagulation and bleeding risk were evaluated by measuring the coagulation parameters PT, APTT, TT, and thromboelastography. The 3-dimensional morphology of platelet aggregates was observed by a microscopic 3-dimensional imaging. Re was a potent inhibitor of SIPA, with an IC 50 of 0.071 mg/mL. It effectively blocked shear stress-induced platelet activation without any significant toxicity. It was highly selective against SIPA, effectively inhibiting vWF-GPIb and the downstream PI3K/Akt signaling pathway. Most importantly, Re did not affect normal blood coagulation and did not increase the risk of bleeding. In conclusion, Re inhibits platelet activation through the inhibition of the vWF-GPIb/PI3K/Akt pathway. Thus, it might be considered as a new antiplatelet drug in the prevention of thrombosis without increasing the risk of bleeding.

Biomechanical activation of blood platelets via adhesion to von Willebrand factor studied with mesoscopic simulations.

Platelet adhesion and activation are essential initial processes of arterial and microvascular hemostasis, where high hydrodynamic forces from the bloodflow impede coagulation. The process relies on von Willebrand factor (VWF)-a linear multimeric protein of blood plasma plays a pivotal role in mechanochemical regulation of shear-induced platelet aggregation (SIPA). Adhesive interactions between VWF and glycoprotein receptors GPIb are crucial for platelet recruitment under high shear stress in fluid. Recent advances in experimental studies revealed that mechanical tension on the extracellular part of GPIb may trigger a cascade of biochemical reactions in platelets leading to activation of integrins [Formula: see text] (also known as GPIIb/IIIa) and strengthening of the adhesion. The present paper is aimed at investigation of this process by three-dimensional computer simulations of platelet adhesion to surface-grafted VWF multimers in pressure-driven flow of platelet-rich plasma. The simulations demonstrate that GPIb-mediated mechanotransduction is a feasible way of platelet activation and stabilization of platelet aggregates under high shear stress. Quantitative understanding of mechanochemical processes involved in SIPA would potentially promote the discovery of new anti-platelet medication and the development of multiscale numerical models of platelet thrombosis and hemostasis.

Water-soluble tomato concentrate modulates shear-induced platelet aggregation and blood flow in vitro and in vivo.

Water-soluble tomato concentrate (WSTC), extracted from mature tomatoes, is the first health product in Europe that has been approved “to help maintain normal platelet activity to maintain healthy blood flow.” We hypothesized that WSTC might exert an influence on blood flow shear stress-induced platelet aggregation (SIPA) and in turn maintains healthy blood flow. We used a microfluidic system to measure the effects of WSTC on SIPA in vitro. We also used the strenuous exercise rat model and the κ-carrageenan-induced rat tail thrombosis model to demonstrate the effects of WSTC on blood flow. WSTC significantly inhibited platelet aggregation at pathological high shear rate of 4,000 s–1 and 8,000 s–1 in vitro (P < 0.05 or P < 0.01). WSTC reduced the platelet adhesion rate and increased the rolling speed of platelets by inhibiting binding to Von Willebrand Factor (vWF) (P < 0.05 or P < 0.01). The oral administration of WSTC for 4 weeks in strenuous exercise rats alleviated hyper-reactivity of the platelets and led to a significant reduction in the plasma levels of catecholamine and IL-6. WSTC treatment also led to a reduction in black tail length, reduced blood flow pulse index (PI) and vascular resistance index (RI), and ameliorated local microcirculation perfusion in a rat model of thrombosis. WSTC exerted obvious inhibitory effects on the platelet aggregation induced by shear flow and alleviated the blood flow and microcirculation abnormities induced by an inflammatory reaction.

Understanding stenosis-induced platelet aggregation on a chip by high-speed optical imaging

Vascular stenosis is a pathological hallmark of atherosclerosis, the leading cause of cardiovascular diseases such as stroke and myocardial infarction. While stenosis-induced thrombus formation has been extensively studied using in vivo and in vitro vascular models, the transient and dynamic process of platelet aggregation remains unclear due to the lack of analytical tools with both high spatial and temporal resolution. Here we report spatiotemporally resolved observation of shear-induced platelet aggregation using an in vitro microfluidic stenosis model and an optical time-stretch imaging system. Specifically, we characterized the size, shape, and population of platelet aggregates at single-cell resolution every 20 min in the presence of different agonists in a 3D stenosis model. Our results indicate a significant enhancement in platelet aggregation when both stenosis and an agonist were present, thus suggesting a synergistic effect of atherogenic blood flow disturbance and circulating factors on platelet activation. In particular, platelets activated by thrombin receptor activator peptide 6 (TRAP-6) led to a broad-sized distribution of platelet aggregates with significantly large aggregates (>240 µm2 in area). These findings are expected to deepen our understanding of the mechanism behind stenosis-induced platelet aggregation and pave the way for development of better antithrombotic therapeutics.

Global Thrombosis Test: Occlusion Is Attributable to Shear-Induced Platelet Thrombus Formation

Herein, we set out a rebuttal to the publication by Claveria and co-workers published in TH Open this month entitled “Global Thrombosis Test: Occlusion by Coagulation or SIPA?” We strongly believe that the conclusions of their paper, suggesting that occlusion (OT) in the Global Thrombosis Test (GTT) is due to coagulation, rather than shear-induced platelet thrombus formation, is incorrect and the evidence and arguments they present are fundamentally flawed, with major errors both in the experimental approach and in the interpretations of the results. The evidence which they demonstrate, shows that occlusion in the GTT is, in fact, caused by high shear induced platelet thrombus formations. We set out herein the evidence for that, based on histology of the thrombus from the GTT in earlier work using electron microscopy showing large platelet aggregates, the very brief timescale of OT in the GTT compared to coagulation time and the sensitivity of the OT in the GTT to the effects of heparin, t-PA and P2Y12 inhibitors. In addition, we revisit the known pathomechanism of high shear-mediated platelet aggregation to underpin our rationale and show that the modifications to the instrument proposed by Claveria and co-authors would render the technique unphysiological. We highlight several methodological concerns and apparent misinterpreted of the data obtained. We present evidence predominantly from the authors’ own data, together with our earlier published data and evidence from the literature, showing that occlusion in the GTT occurs do to shear-induced platelet aggregation.

Inhibition of the Shear-Induced Platelet Microparticle Formation by a mAb Against GPIb alpha Is Associated with Protein Tyrosine Dephosphorylation and the Blockade of Filamin Cleavage.

Platelet microparticle (MP) formation during shear-induced platelet aggregation (SIPA) contributes to a relevant part of thrombin generation. A monoclonal antibody (mAb) Ib-23 against platelet GPIb alpha (provided by B.S.) inhibits von Willebrand factor binding, ristocetin-induced platelet aggregation (RIPA), shear induced MP formation and thrombin generation. Platelet calpain affects several functions including MP-formation. One of several target substrates of calpain, filamin, binds to the cytoplasmic tail of GPIbalpha and mediates the assembly of the contractile elements during platelet activation. We therefore hypothesized that the inhibitory mechanism of our mAb Ib-23 on MP formation is a “calpeptin-like effect”, i.e. an inhibition of calpain affecting the protein tyrosine phosphorylation.PPACK-anticoagulated platelet-rich plasma or washed platelets (250′000/μl) were exposed to a shear rate of 5000 sec−1 for 5 min in a cone-plate viscometer. The mAb Ib-23 was added at the RIPA-inhibiting IC80–90. The samples were fluorescence-labelled with an anti-GPIIb-PE mAb to monitor SIPA and MP formation and analyzed by flow cytometry. Resting and sheared platelets were lysed in SDS-PAGE loading buffer for the analysis of filamin degradation and protein tyrosine phosphorylation.Results: Epitope mapping showed that mAb Ib-23 binds between residues 234 and 456 on GP Ib alpha. Exposure of platelets to high shear rates induced the activation of calpain as demonstrated by the degradation of filamin, a phenomenon blocked by the mAb Ib-23. Ib-23 did not inhibit the SIPA induced tyrosine phosphorylation of high molecular weight proteins like FAK, Syk and alpha-actinin, it even weakly enhanced the tyrosine phosphorylation of these proteins. Moreover, Ib-23 prevented the dephosphorylation of low molecular weight proteins (appr. 40 and 33kDa). Annexin V-binding was unaffected by Ib-23. In contrast, abciximab, a GP IIb/IIIa antagonist, partially inhibited SIPA and annexin binding, but not MP generation. Abciximab weakly inhibited the degradation of filamin, inhibited SIPA-induced tyrosine phosphorylation of FAK and Syk, but did not prevent the tyrosine dephosphorylation of the low molecular weight proteins. Control-experiments with calpeptin demonstrated a similar inhibition of filamin degradation as with mAb Ib-23. An isotype control antibody was without effects. The intact antibody Ib-23 was more effective than the Fab2 fragment. A mAb against the FcgammaRII receptor (IV.3, to exclude a bridging of the FcgammaRII and GpIbalpha via the antibody's Fc part) did not affect MP-formation.We conclude that binding of mAb Ib-23 on the outside of GP Ib alpha inhibits SIPA-induced MP formation and thrombin generation possibly via a “calpeptin-like effect” which inhibits the activation of calpain and of protein tyrosine phosphatases.

Application of a Simple Microfluidic Chip Analysis Technology to Evaluate the Inhibitory Role of Protocatechuic Acid on Shear-Induced Platelet Aggregation.

This study aimed to develop a simple microfluidic chip analysis technology to study the inhibitory effect of protocatechuic acid on shear-induced platelet aggregation. The microfluidic chip designed in this study simulates 80% fixed narrow microchannels. This microchannel narrow model uses the finite element analysis module of the three-dimensional modeling software solidwork to analyze fluid dynamic behavior. Blood treated with protocatechuic acid at 1, 2, 4, 8, or 16 µg/mL was passed through the microchannel stenosis model at a shear rate of 10,000 s−1. The platelet adhesion and aggregation behaviors were then measured using fluorescence microscopy and observed in real time. Simultaneously, the antiplatelet aggregation effect of protocatechuic acid was analyzed using thromboelastography and photoelectric turbidimetry. The designed stenosis model of the microfluidic chip can produce a gradient of fluid shear rate, and the gradient of fluid shear rate can induce platelet aggregation. Under this model, the degree of platelet adhesion and aggregation increased as the shear rate increased. In the experimental concentration range of 0–8 µmol/mL, protocatechuic acid exerted a concentration-dependent inhibition of platelet aggregation. In contrast, thromboelastography and photoelectric turbidimetry failed to demonstrate an inhibitory effect. The microfluidic chip analysis technology developed in this study can be used to study the effect of protocatechin in inhibiting platelet aggregation induced by shear rate in vitro. This technology is simple to operate and can be used as a new type of antiplatelet aggregation analysis technology for screening studies of novel potential antiplatelet aggregation drugs.

Inhibition of Shear-Induced Platelet Aggregation by Xueshuantong via Targeting Piezo1 Channel-Mediated Ca2+ Signaling Pathway.

XueShuanTong (XST) comprising therapeutically active ginsenosides, a lyophilized extract of Panax notoginseng roots, is extensively used in traditional Chinese medicine to treat ischemic heart and cerebrovascular diseases. Our recent study shows that treatment with XST inhibits shear-induced thrombosis formation but the underlying mechanism remained unclear. This study aimed to investigate the hypothesis that XST inhibited shear-induced platelet aggregation via targeting the mechanosensitive Ca2+-permeable Piezo1 channel by performing platelet aggregation assay, Ca2+ imaging and Western blotting analysis. Exposure to shear at physiologically (1,000–2000 s−1) and pathologically related rates (4,000–6,000 s−1) induced platelet aggregation that was inhibited by treatment with GsMTx-4. Exposure to shear evoked robust Ca2+ responses in platelets that were inhibited by treatment with GsMTx-4 and conversely enhanced by treatment with Yoda1. Treatment with XST at a clinically relevant concentration (0.15 g L−1) potently inhibited shear-induced Ca2+ responses and platelet aggregation, without altering vWF-mediated platelet adhesion and rolling. Exposure to shear, while resulting in no effect on the calpain-2 expression in platelets, induced calpain-2-mediated cleavage of talin1 protein, which is known to be critical for platelet activation. Shear-induced activation of calpain-2 and cleavage of talin1 were attenuated by treatment with XST. Taken together, our results suggest that XST inhibits shear-induced platelet aggregation via targeting the Piezo1 channel to prevent Piezo1-mediated Ca2+ signaling and downstream calpain-2 and talin1 signal pathway, thus providing novel insights into the mechanism of the therapeutic action of XST on platelet aggregation and thrombosis formation.

Mechanobiology of shear-induced platelet aggregation leading to occlusive arterial thrombosis: A multiscale in silico analysis

Occlusive thrombosis in arteries causes heart attacks and strokes. The rapid growth of thrombus at elevated shear rates (~10,000 1/s) relies on shear-induced platelet aggregation (SIPA) thought to come about from the entanglement of von Willebrand factor (VWF) molecules. The mechanism for SIPA is not yet understood in terms of cell- and molecule-level dynamics in fast flowing bloodstreams. Towards this end, we develop a multiscale computational model to recreate SIPA in silico, where the suspension dynamics and interactions of individual platelets and VWF multimers are resolved directly. The platelet-VWF interaction via GP1b-A1 bonds is prescribed with intrinsic binding rates theoretically derived and informed by single-molecule measurements. The model is validated against existing microfluidic SIPA experiments, showing good agreement with the in vitro observations in terms of the morphology, traveling distance and capture time of the platelet aggregates. Particularly, the capture of aggregates can occur in a few milliseconds, comparable to the platelet transit time through pathologic arterial stenotic sections and much shorter than the time for shear-induced platelet activation. The multiscale SIPA simulator provides a cross-scale tool for exploring the biophysical mechanisms of SIPA in silico that are difficult to access with single-molecule measurements or micro-/macro-fluidic assays only.

Microfluidic assay of antiplatelet agents for inhibition of shear-induced platelet adhesion and activation.

We have developed a microfluidic system to perfuse whole blood through a flow channel with an upstream stenotic region and a downstream protein capture region. This flow-based system was used to assay how effectively antiplatelet agents suppress shear-induced platelet adhesion and activation downstream of the stenotic region. Microcontact printing was used to covalently attach one of three platelet binding proteins [fibrinogen, collagen, or von Willebrand factor (vWf)] to the surface of the downstream capture region. Whole blood with an antiplatelet agent was transiently exposed to an upstream high wall shear rate (either 4860 s-1 or 11 560 s-1), and subsequently flowed over the downstream capture region where the platelet adhesion was measured. Several antiplatelet agents (acetylsalicylic acid, tirofiban, eptifibatide, anti-vWf, and anti-GPIbα) were evaluated for their efficacy in attenuating downstream adhesion. Following antibody blocking of vWf or GPIbα, downstream platelet activation was also assessed in perfused blood by flow cytometry using two activation markers (active GPIIb/IIIa and P-selectin). Acetylsalicylic acid demonstrated its inability to diminish shear-induced platelet adhesion to all three binding proteins. GPIIb/IIIa inhibitors (tirofiban and eptifibatide) significantly reduced platelet adhesion to fibrinogen. Antibody blocking of vWf or GPIbα effectively diminished platelet adhesion to all three capture proteins as well as platelet activation in perfused blood, indicating an essential role of vWf-GPIbα interaction in mediating shear-induced platelet aggregation.

Interferon Alpha Therapy Increases Pro-Thrombotic Biomarkers in Patients with Myeloproliferative Neoplasms.

Myeloproliferative neoplasms (MPN) are associated with an increased risk of arterial and venous thrombosis. Pegylated-interferon alpha (IFN) and hydroxyurea (HU) are commonly used to treat MPN, but their effect on hemostasis has not yet been studied. The aim of our study was to determine whether IFN and HU impact the biological hemostatic profile of MPN patients by studying markers of endothelial, platelet, and coagulation activation. A total of 85 patients (50 polycythemia vera and 35 essential thrombocythemia) were included: 28 treated with IFN, 35 with HU, and 22 with no cytoreductive drug (non-treated, NT). Von Willebrand factor, shear-induced platelet aggregation, factor VIII coagulant activity (FVIII:C), fibrinogen, and thrombin generation with and without exogenous thrombomodulin were significantly higher in IFN-treated patients compared to NT patients, while protein S anticoagulant activity was lower. In 10 patients in whom IFN therapy was discontinued, these hemostatic biomarkers returned to the values observed in NT patients, strongly suggesting an impact of IFN therapy on endothelial and coagulation activation. Overall, our study shows that treatment with IFN is associated with significant and reversible effects on the biological hemostatic profile of MPN patients. Whether they could be associated with an increased thrombotic risk remains to be determined in further randomized clinical studies.

Shear-induced platelet aggregation: 3D-grayscale microfluidics for repeatable and localized occlusive thrombosis.

Atherothrombosis leads to complications of myocardial infarction and stroke as a result of shear-induced platelet aggregation (SIPA). Clinicians and researchers may benefit from diagnostic and benchtop microfluidic assays that assess the thrombotic activity of an individual. Currently, there are several different proposed point-of-care diagnostics and microfluidic thrombosis assays with different design parameters and end points. The microfluidic geometry, surface coatings, and anticoagulation may strongly influence the precision of these assays. Variability in selected end points also persists, leading to ambiguous results. This study aims to assess the effects of three physiologically relevant extrinsic design factors on the variability of a single end point to provide a quantified rationale for design parameter and end-point standardization. Using a design of experiments approach, we show that the methods of channel fabrication and collagen surface coating significantly impact the variability of occlusion time from porcine whole blood, while anticoagulant selection between heparin and citrate did not significantly impact the variability. No factor was determined to significantly impact the mean occlusion time within the assay. Occlusive thrombus was found to consistently form in the first third (333 μm) of the high shear zone and not in the shear gradient regions. The selection of these factors in the design of point-of-care diagnostics and experimental SIPA assays may lead to increased precision and specificity in high shear thrombosis studies.

Leu33Pro (PlA) polymorphism of integrin beta3 modulates platelet Src pY418 and focal adhesion kinase pY397 phosphorylation in response to abnormally high shear stress.

Shear stress can activate platelet integrin-mediated signaling that leads to shear-induced platelet aggregation (SIPA) and eventually contribute to acute myocardial infarction. The major platelet integrin αIIbβ3 is polymorphic at residue 33 [Leu33Pro (PlA) polymorphism]. The Pro33 isoform has been shown to have a prothrombotic phenotype. In this work, we studied the impact of Leu33/Pro33 polymorphism on the shear-induced integrin-mediated Src and FAK activation in platelets. Platelets of both genotypes were placed on immobilized fibrinogen or heat activated BSA and were exposed to physiological (500/s) or abnormally high (5000/s) shear rates for 2-10 min. Platelets after exposure to shear were analysed for Src pY418 and FAK pY397 activities. Whereas physiological shear stress does not affect platelet signaling, abnormally high-shear stress considerably elevates Src and FAK phosphorylation in both Pro33 and Leu33 platelets. Both under static and flow conditions, Pro33 platelets exhibited a significantly higher Src and FAK activities than Leu33 platelets. Interestingly, even in the absence of the αIIbβ3-fibrinogen interaction, we could detect a shear-induced integrin-mediated signaling of Src and FAK in platelets. In parallel experiments in which platelets were pretreated with abciximab, an integrin αIIbβ3 antagonist, activation of both kinases by shear was inhibited. Taken together, our data indicates an important role of αIIbβ3 and shows that Leu33Pro polymorphism modulates the integrin-mediated Src and FAK signaling in platelets in response to shear stress.

Impact of shear stress on Src and focal adhesion kinase phosphorylation in fibrinogen-adherent platelets

: Shear stress alone can activate platelets resulting in a subsequent platelet aggregation, so-called 'shear-induced platelet aggregation'. In our work, we analyzed how differently elevated shear stress impacts the Src and focal adhesion kinase (FAK) activation in fibrinogen-adherent human platelets. We detected the extents of Src pY418 and FAK pY397 activations in platelets on immobilized fibrinogen and over BSA under shear conditions. Moreover, we analyzed the role of αIIbβ3 in the shear-induced platelet signaling by performing our experiments in the presence of the αIIbβ3-antagonist Abciximab. Abnormally high shear rates (5000 s) significantly increased the extent of phosphorylation of both tyrosine kinases after short (2 min) incubation time independently of the presence or absence of the integrin αIIbβ3 ligand, fibrinogen. We could see considerably greater Src activation on immobilized fibrinogen than on BSA, but the extent of FAK Y397 phosphorylation was independent on the matrix. Abciximab not only reduced the Src and FAK signaling in platelets exposed to 5000 s on immobilized fibrinogen, but in platelets exposed to 5000 s over BSA as well. Our data indicate that whereas Src activation under shear stress is dominantly ligand-dependent, FAK signaling seems to be mostly shear induced.

Pro-Coagulant and Pro-Inflammatory Effect of Interferon Alpha in Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPN) are associated with an increased risk of arterial and venous thrombosis. Besides clinical factors (previous history of thrombosis, age > 60 years, cardiovascular risk factors), procoagulant phenotype, proadhesive properties of the endothelium and secretion of inflammatory cytokines, contribute to the pathogenesis of thrombosis in MPN. Pegylated interferon alpha (IFN) has obviously a great therapeutic efficacy on hematopoietic cell proliferation but its other biological effects particularly on hemostasis and inflammation have not been determined.The aim of the study was to determine whether IFN impacts the biological profile including endothelial and platelet markers compared to MPN patients treated by hydroxyurea (HU) and non-treated (NT) patients.Patients with polycythemia vera (PV) or essential thrombocythemia (ET) treated with IFN or HU or without any cytostatic drug were included. Platelet membrane glycoproteins and platelet activation were measured by flow cytometry. We evaluated shear-induced platelet aggregation (SIPA) at 4000 sec-1 that mimics stenotic arteries. We measured coagulation proteins by their activity and endothelial parameters (von Willebrand factor (VWF) antigen and activity, soluble thrombomodulin). Thrombin generation was measured in platelet poor plasma and platelet rich plasma. Platelet accumulation on immobilized collagen was measured using whole blood perfused at 1500 sec-1 in flow chambers. Statistical analysis was performed by ANOVA to compare the 3 groups of patients (p<0.05) and post-test Newman-Keuls to compare groups side-by-side (p<0.01).Eighty-eight patients were included: 28 treated by IFN (20 PV and 8 ET), 38 treated by HU (27 PV and 11 ET) and 22 NT (6 PV and 16 ET). All patients were treated with aspirin 75 or 100 mg/day. Prior history of thrombosis occurred in 25.7% of ET and in 31.5% of PV. During the follow up (> 6months), only 1 arterial and 2 venous thrombosis were observed.Hematological parameters and JAK2 V617F status was in the expected range in ET and PV patients.Surprisingly, we observed some significant effect of IFN on 1) platelets with a decreased GPVI expression compared to HU- and non-treated patients and increased levels of SIPA at 4000 sec-1. 2) endothelial cells with significantly increased levels of Willebrand antigen and activity. 3) coagulation parameters with decreased protein S activity and increased levels of factor VIII:C and fibrinogen (table 1). All the other parameters tested were similar in the 3 groups of patients.We had the opportunity to test 10 patients at least 6 months after IFN discontinuation. IFN was stopped for side effects in 6 patients, for complete molecular response in 2 and for normalization of hematological parameters in 2 others. GPVI expression, SIPA, VWF activity and antigen, protein S activity, FVIIIC and fibrinogen returned to levels similar to those of non-treated patients and were significantly different from the levels in IFN-treated patients (table 2). These results indicate that IFN was responsible for these biological modifications.In conclusion, these unexpected data showing an increase of procoagulant and inflammatory markers in IFN-treated MPN patients could be considered as a part of IFN biological activity. Though minimal, these alterations could require more intensive preventive anticoagulation than aspirin in patients with heterozygous FV Leiden or FII G20210A mutation. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Platelet-Delivered Recombinant ADAMTS13 Inhibits Platelet Adhesion and Aggregation on Collagen Surface Under Flow in the Presence of Autoantibodies from Acquired Thrombotic Thrombocytopenic Purpura

Background: Acquired thrombotic thrombocytopenic purpura (TTP) is a rare but potentially fatal syndrome. It is primarily caused by severe deficiency of plasma ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) activity. ADAMTS13 is a plasma metalloprotease that cleaves von Willebrand factor (VWF), thereby reducing the platelet adhesion and aggregation under flow. Plasma exchange is the only effective initial therapy for acquired autoimmune TTP. However, in hospital mortality rate remains as high as 10-20% in addition to the concern about the complications associated with the placement of central catheter and the use of plasma products. Therefore, a better and more effective therapy is urgently needed.Objectives: To develop a more effective therapy, we determined the uptake and package of recombinant ADAMTS13 (rA13) in platelets and assessed the efficacy of platelets-delivered rA13 in inhibiting platelet adhesion and aggregation and thrombus formation under arterial shear.Methods: Human platelets were isolated from whole blood, washed, and incubated with various concentrations of rA13 (1-100 microgram/ml) at 25 and 37 degree Celsius for various times. The amount of rA13 uptake by platelets was determined in cell lysate by Western blotting and FRETS-VWF73 assays and in fixed cells by immunofluoresent microscopy and flow cytometry. The function of platelet delivered rA13 was also determined by shear-induced platelet adhesion and aggregation on collagen surface using a microfluidic system.Results: Freshly isolated and blood bank stored platelets were able to update rA13 in a temperature, concentration, and time-dependent manner. When rA13 (5 microgram/ml) was incubated with platelets, nearly all platelets were positive for rA13, assessed by immunofluoresent microscopy and flow cytometry. The rA13 inside platelets remained intact and was proteolytically active in cleaving FRETS-VWF73. Confocal imaging revealed that rA13 was partially co-localized with VWF in alpha granules of platelets. Microfluidic assay demonstrated that platelet-delivered rA13 was able to dramatically inhibit platelet adhesion and aggregation on collagen-coated surface under arterial shear (100 dyne/cm2) in the absence and presence of a human monoclonal antibody against ADAMTS13 (scFv4-20) that was isolated from a patient with acquired autoimmune TTP. These results were consistent with the inhibitory effects observed with mouse transgenic platelets expressing rA13 under the same conditions. When 1/3 of whole blood Adamts13-/- platelets were replaced with transgenic platelets (containing rA13), the coverage of all fluorescent platelets onto a VWF-collagen surface was dramatically reduced (data not shown).Conclusion: Our results demonstrate that platelets uptake and deliver rA13 to the site of thrombus formation under flow and the platelet-delivered rA13 may be efficacious for treating acquired TTP with inhibitors. DisclosuresZheng:Ablynx: Consultancy; Alexion: Research Funding.

A simple method for activating the platelets used in microfluidic platelet aggregation tests: Stirring-induced platelet activation.

High-shear stimulation is well known as one of the key factors affecting platelet activation and aggregation, which can lead to the formation of a thrombus. In one of our previous studies, we introduced migration distance-based platelet function analysis in a microfluidic system. In this study, we set out to examine the effects of stirring on shear-induced platelet activation and aggregation in a chamber system by using a rotating stirrer. We found that the rotating stirrer caused not only rotational shear flow but also a strong radial secondary flow. The latter flow led to efficient mixing in the chamber. Moreover, the rotational flow led to the generation of shear stress, the magnitude of which can be controlled to activate the platelets. Activated platelets tend to aggregate themselves. The maximum platelet aggregation was observed at a critical shear rate of 3100 s−1, regardless of the stirrer shape. Furthermore, the time taken to attain maximum aggregation was significantly shortened when using a wide stirrer (30 s) instead of a narrow one (180 s). When using a flat stirrer, the non-uniform shear field in the chamber system was resolved with the radial secondary flow-induced mixing; thus, most of the platelets were homogenously activated. The stirring-induced platelet activation mechanism was experimentally confirmed in a microfluidic system for a platelet aggregation test while monitoring the migration distance until the microfluidic channel is occluded. Our findings indicate that the present system, consisting of a rotating stirrer and a confined chamber, provides effective shear stimulation for activating platelets and inducing platelet aggregates.